Streptococcus agalactiae has emerged as a major cause of neonatal sepsis and late-onset meningitis in infants. Despite the sensitivity of this organism to antibiotics, the high attack rate and rapid onset of disease results in high morbidity (50%) and mortality (20%). The polysaccharide bacterial capsule of group B Streptococcus (GBS) is well studied and plays an important role in both virulence and immunity. However, vaccines prepared from these capsular antigens are not fully protective in humans. A family of polypeptides, called C proteins, has been described in GBS that appears to play a role in both virulence and immunity. To identify and characterize proteins that are involved in immunity and virulence of GBS, a recombinant DNA library of chromosomal DNA from GBS was prepared in E. coli. A plasmid-based expression vector was developed for cloning bacterial chromosomal DNA that has several advantages over libraries constructed with gt11. This library is being screened for expression of GBS proteins with antisera to GBS proteins that have been shown to be protective in a mouse lethality model. Five genes have been isolated form GBS whose gene products react with protective antisera prepared against GBS proteins. Interestingly, four of these gene products do not appear to be present in GBS strains that do not carry the C proteins. An animal model is used to determine if the cloned gene products elicit protective antibodies. Those GBS proteins that elicit protective antibodies are, by definition, C proteins. Preliminary biochemical and immunological characterization of the gene products of the clones suggests that at least two genes encoding C proteins have been isolated. The overall goals of this project are to identify and characterize the structure, function, regulation and expression of genes associated with the C protein(s). To determine the role of these genes in pathogenesis, isogenic strains of GBS will be prepared with these genes deleted. The immunity and virulence of these mutants will be evaluated in an animal model. C proteins expressed from the cloned genes will be isolated and their role in immunity studied. The long term goal is to study the potential of these proteins to serve as a polypeptide backbone for GBS capsular polysaccharides and to develop an effective conjugate vaccine against GBS.